基于多重连接探针扩增技术的食品中六重过敏原成分检测

Multiplex Ligation-Dependent Probe Amplification for Simultaneous Detection of Six Allergenic Ingredients in Foods

基于多重连接探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术建立了一种六重过敏原成分检测方法,可实现食品中大豆、芝麻、花生、杏仁、榛子和核桃6种成分的同时检测。选取多拷贝ITS基因为靶标基因,设计并合成6组特异性杂交探针。探针经杂交、连接和聚合酶链式反应得到扩增片段,经毛细管电泳分析扩增片段大小可明确区分6种过敏原成分。该体系经20余种相关植物、动物及微生物DNA验证显示其特异性良好,经模拟参考样品验证其检出限为5 mg/kg。30份不同种类市售食品MLPA检测结果表明,该方法性能满足实际样品的过敏原的多重检测。因此,本研究建立的基于MLPA技术的六重过敏原检测方法特异性强、灵敏度高,能够为食品过敏原评估、标识管理和风险控制提供技术支撑。

A multiplex ligation-dependent probe amplification(MLPA) technique was developed for simultaneous detection of six food allergens: soy, sesame, peanut, almond, hazelnut and walnut. Six pairs of species-specific MLPA probes targeting the internal transcribed spacer(ITS) sequences were designed and synthesized. The probes were hybridized with the target DNA, ligated and amplified by polymerase chain reaction(PCR) and the amplicons were detected using capillary electrophoresis, revealing that they could clearly discriminate the six food allergens. Good specificity of the MLPA system was observed for more than 20 plants, animals and bacteria. The limit of detection(LOD) of the assay was determined to be 5 mg/kg for model cookie spiked with different concentrations of the target species. The applicability of the MLPA assay was demonstrated through the analysis of 30 different commercial products, evidencing good performance in practical applications. Therefore, the MLPA technique is highly specific and sensitive and can provide technical support for the assessment, label management and risk control of food allergens.